Effect of storage on the nitro blue tetrazolium reduction test in dog blood samples.

INTRODUCTION: The nitro blue tetrazolium (NBT) reduction test (NBTT) has been used for measuring the metabolic activity of phagocytes of mammals. Activated neutrophils transform NBT into formazan in the cytoplasm. The NBTT can detect the activation of neutrophils in peripheral blood and is used to assess neutrophil function in dogs. However, the NBTT is not used frequently in the clinical setting, as samples should be processed after blood collection. OBJECTIVE: The aim of this study was to evaluate the effect of storage on NBTT in dog blood samples. MATERIALS AND METHODS: Residual EDTA blood samples from 22 dogs were included of different ages, breeds, and sex. The buffy coat layer was separated from the blood and incubated with 0.1% NBT. The NBTT was performed at 0, 24, 48, and 72 h after the collection of blood. Blood samples were stored at 4°C until the tests were performed. Blood smears were evaluated by light microscopy, and the NBT reduction rate was reported, which represents the percentage of activated neutrophils. The NBT reduction rate was calculated after counting 300 neutrophils in each slide. RESULTS: The means of NBTT in dog blood samples at 0, 24, 48, and 72 h were 8.3%, 8.5%, 8.7%, and 7.8%, respectively. No significant differences were observed between time points. CONCLUSIONS: This study showed that the NBTT can be performed up to 72 h after the collection of canine blood if correctly refrigerated at 4°C. This finding supports the performance of the NBTT in the clinical setting.

Serological survey of Leishmania infantum in apparently healthy dogs in different areas of Spain.

BACKGROUND: Canine leishmaniosis caused by Leishmania infantum is an endemic disease in Spain. The dog is considered the main reservoir, and the detection of specific serum antibodies against L. infantum antigen is the most used technique for diagnosing this infection. The LEISCAN LEISHMANIA ELISA test is a commercialized enzyme-linked immunosorbent assay for the detection and measurement of canine anti-Leishmania serum antibodies. OBJECTIVES: The aim of this study was to assess seroprevalence results of apparently healthy dogs in different areas of Spain using LEISCAN. METHODS: Collection of sera from 5451 apparently healthy dogs was performed between 2020 and 2021 in different areas of Spain. Dogs were of adult age (≥12 months), were not previously diagnosed with clinical leishmaniosis or vaccinated against Leishmania and did not present clinical signs compatible with L. infantum infection. LEISCAN was performed following the manufacturer's protocol. RESULTS: The overall seroprevalence was 5.5%. The highest seroprevalences were found in the Southeast of Spain: Comunidad Valenciana (14%) and Región de Murcia (14%), whereas the lowest seroprevalences were found in Northern Spain: Galicia (1%), Navarra (2%) and Castilla y León (2%) (p-value <0.001). CONCLUSIONS: In conclusion, the seroprevalence for L. infantum in apparently healthy dogs in Spain varied from almost no infection to being over 10%.

Exploring the Relationship between Neutrophil Activation and Different States of Canine L. infantum Infection: Nitroblue Tetrazolium Test and IFN-γ.

This study aimed to investigate the role of neutrophils in canine leishmaniosis by assessing neutrophil activation and its relationship with different states of L. infantum infection and antibody and IFN-γ production. Dogs were categorized into five groups: healthy-seronegative (n = 25), healthy-seropositive (n = 21), LeishVet-stage I (n = 25), Leishvet-stage II (n = 41), and LeishVet-stage III-IV (n = 16). Results of the nitroblue tetrazolium reduction test (NBT) showed significantly higher neutrophil activation in stage I (median:17.17, range: [7.33-31.50]%) compared to in healthy-seronegative (4.10 [1.20-18.00]%), healthy-seropositive (7.65 [3.98-21.74]%), stage II (6.50 [1.50-28.70]%), and stage III-IV (7.50 [3.00-16.75]%) groups (p < 0.0001). Healthy-seropositive dogs also displayed higher values than all groups except stage I. Stages II and III-IV did not show significant differences compared to healthy-seronegative. Regarding IFN-γ, stage I dogs had higher concentrations (median:127.90, range: [0-3998.00] pg/mL) than healthy-seronegative (0 [0-109.50] pg/mL) (p = 0.0002), stage II (9.00 [0-5086.00] pg/mL) (p = 0.045), and stage III-IV (3.50 [80.00-548.80] pg/mL) (p = 0.02) dogs. Stage II dogs showed increased IFN-γ compared to healthy-seronegative dogs (p = 0.015), while stage III-IV dogs had no significant differences compared to healthy-seronegative dogs (p = 0.12). Healthy-seropositive dogs had elevated IFN-γ concentrations compared to healthy-seronegative dogs (p = 0.001) and dogs in stage III-IV (p = 0.03). In conclusion, neutrophil activation was higher in dogs with mild disease and healthy-seropositive dogs, and a relationship between neutrophil activation and the production of IFN-γ was found.

A blinded, randomized and controlled multicenter clinical trial to assess the efficacy and safety of Leisguard® as an immunotherapeutic treatment for healthy Leishmania infantum-seropositive dogs.

BACKGROUND: Domperidone (Leisguard®) is an immunomodulatory drug used as a preventive measure in healthy dogs. However, no studies have been published in healthy Leishmania infantum-seropositive dogs. The aim of this study was to evaluate the clinical efficacy and safety of domperidone as immunotherapy in Leishmania-seropositive healthy dogs. METHODS: Sixty-seven dogs were treated with domperidone at 0.5 mg/kg and 44 dogs received placebo, once daily for 4 consecutive weeks. Monthly treatments were repeated every 4 months until the end of the 1-year follow-up period. Veterinary examinations were performed on days 0, 30, 120, 150, 240, 270 and 360. Samples of blood and urine were collected on days 0, 120, 240 and 360 for routine laboratory tests and quantitative in-house ELISA for the detection of L. infantum-specific antibodies. Furthermore, Leishmania real-time PCR and IFN-γ ELISA were performed at day 0 and the end of the study. Dogs that developed disease were withdrawn from the study and classified as sick dogs. Adverse drug reactions were reported. RESULTS: Thirty dogs developed disease during the follow-up period: 13/67 (19.4%) in the group treated with domperidone and 17/44 (38.6%) in the placebo-treated group (P = 0.03). Low-seropositive dogs treated with domperidone (4/40, 9.1%) were significantly less likely to develop disease compared to low-seropositive dogs treated with placebo (7/24, 29.2%; P = 0.04), while no differences were found between domperidone (9/23, 39.1%) and placebo (10/20, 50%) in medium- to high-seropositive dogs. At the end of the study, a higher proportion of Leishmania PCR-positive dogs was observed in the placebo-treated group (16/33, 48.5%) compared to the domperidone group (13/51, 25.5%; P = 0.04). Furthermore, low-seropositive dogs treated with domperidone with an increase of IFN-γ concentration presented a higher increase than those treated with placebo at the end of the study. Four dogs treated with domperidone presented self-limiting diarrhea. CONCLUSIONS: Healthy dogs with low L. infantum antibody levels treated with domperidone were less likely to develop disease compared to placebo-treated dogs. Furthermore, domperidone presented a good safety profile.

Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs.

Canine leishmaniosis caused by Leishmania infantum is a disease with a wide range of clinical manifestations. Epidemiological serosurveys performed in Europe often lack a thorough assessment of clinical health status of studied dogs. The aim of this study was to evaluate signalment, immunological and parasitological status and clinicopathological findings of L. infantum-seropositive apparently healthy dogs (n = 212) living in endemic areas. Routine laboratory tests, endpoint in-house ELISA to quantify the anti-Leishmania antibodies, blood Leishmania qPCR and IFN-γ ELISA were performed. All dogs enrolled were L. infantum-seropositive and were classified as healthy (n = 105) or sick (n = 107) according to LeishVet guidelines. The sick group presented a higher proportion of medium to high antibody levels and positive qPCR and lower IFN-γ concentration compared to the healthy group. Sick dogs were mostly classified in LeishVet stage IIa. Biochemical alterations (98%) were the most common clinicopathological findings, with fewer urinary tract (46%) and hematological (40%) alterations. Apparently healthy L. infantum-seropositive dogs can be classified between truly healthy dogs and sick dogs with clinicopathological findings. Sick dogs presented medium to high seropositivity and parasitemia and low IFN-γ concentrations, and their most common clinicopathological abnormalities were serum protein alterations followed by proteinuria and lymphopenia.

Use of preventive measures and serological screening tools for Leishmania infantum infection in dogs from Europe.

BACKGROUND: There are several screening tools for detecting Leishmania infantum infection in dogs and various preventive measures to protect against it. Some studies have investigated them, but not many have described their current use. The aim of this study was to investigate which preventive measures and serological screening tools for L. infantum infection were employed from 2012 to 2018 in dogs from different endemic European countries. METHODS: A set of electronic datasheets was completed for each dog from several veterinary centres. Classification of preventive measures included: (1) repellents, (2) vaccines and (3) immunomodulators. Classification of serological tests included the: (1) direct agglutination test (DAT), (2) enzyme-linked immunosorbent assay (ELISA), (3) indirect immunofluorescence (IFI), (4) rapid tests and (5) other assays. Dogs were also classified depending on their risk of exposure and living area. RESULTS: Information from 3762 dogs was gathered. Preventive measures were applied in 91.5% of dogs and the most frequently used were repellents (86.2%) followed by vaccines (39.8%) and Leisguard® (15.3%). The different types of repellents (collar and spot-on) were used similarly. A combination of a vaccine and repellents was preferred in the high-risk group while the low-risk preferred a combination of Leisguard® and a repellent (Chi-square test: X2 = 88.41, df = 10, P < 0.001). Furthermore, all preventive measures were similarly used through the years except for repellents, which were predicted to have a small increase of use each year. Regarding serological screening tools, the most used were rapid and ELISA tests. Rapid tests, ELISA tests and DAT were used similarly through the years, but a significant change was found in the use of IFI and other assays whose use decreased a little each year. CONCLUSIONS: Repellents were the preferred measure, while vaccines and Leisguard® were second-line options. Some dogs were not treated by any measures, which highlights the need for dog owner education. Moreover, there seems to be a preference for rapid tests in the clinical setting to detect specific L. infantum antibodies while ELISA or IFI are less often employed. This underlines an increasing problem, as qualitative rapid tests have a variable diagnostic performance limiting the adequate diagnosis of seropositive dogs in endemic areas.

Detection of specific antibodies against Leishmania infantum in canine serum and oral transudate using an in-house ELISA.

BACKGROUND: Canine leishmaniosis caused by the protozoan Leishmania infantum is a complex infection due to its variable clinical signs and laboratory findings. Therefore, a broad range of techniques is available for diagnosis. Testing for specific antibodies in serum is the most commonly used technique, although the testing of other body fluids, such as oral transudate (OT), can be an alternative as its collection is non-invasive and testing can be performed by untrained personnel. The aim of this study was to assess and compare the detection of L. infantum-specific antibodies in paired samples of serum and OT collected from apparently healthy dogs and dogs with clinical leishmaniosis using an in-house enyzme-linked immunosorbent assay (ELISA). METHODS: Serum and OT were collected from 407 dogs, which varied in breed, sex, age, lifestyle and clinical status, by many practicing veterinarians in Spain. The main geographical areas of sampling included Barcelona (n = 110), Mallorca (n = 94), Cadiz (n = 54) and Asturias (n = 47). The majority of infected dogs were apparently healthy (89.9%) while 41 presented clinical signs and/or clinicopathological abnormalities compatible with L. infantum infection and subsequently diagnosed with leishmaniosis (10.1%). An in-house ELISA was performed to quantify the anti-Leishmania antibodies in serum and OT. RESULTS: The L. infantum infection rate determined by the in-house ELISA was 37.1% in serum samples and 32.7% in OT samples. Serum and OT ELISA results showed a positive correlation (Spearman's correlation coefficient rs = 0.6687, P < 0.0001). The percent agreement between the serum and OT ELISA results was 84%, while agreement according to Cohen's kappa statistic (κ) was substantial (0.66) when all samples were analyzed. The highest percent agreement (92.1%) between both tests was found in dogs from low endemicity regions and from sick dogs, with both groups presenting almost perfect agreement according to Cohen's κ agreement test (0.84). Few seronegative dogs (n = 23) tested positive by the OT ELISA. The agreement between serum and OT went from almost perfect to moderate when the geographical distribution and clinical status were analyzed. CONCLUSIONS: The results of this study demonstrated an almost perfect to moderate agreement between OT and serum samples tested using the in-house ELISA. These results are particularly promising in sick dogs with high antibody levels while the results seem less optimal in apparently healthy dogs with low antibody levels.

Prospective serological and molecular cross-sectional study focusing on Bartonella and other blood-borne organisms in cats from Catalonia (Spain).

BACKGROUND: There is limited clinical or epidemiological knowledge regarding Bartonella infection in cats, and no serological studies have compared the presence of antibodies against different Bartonella species. Moreover, there are limited feline Bartonella studies investigating co-infections with other vector-borne pathogens and the associated risk factors. Therefore, the objective of this study was to investigate Bartonella spp. infections and co-infections with other pathogens in cats from Barcelona (Spain) based on serological and/or molecular techniques and to determine associated risk factors. METHODS: We studied colony and owned cats (n = 135). Sera were tested for Bartonella henselae-, Bartonella quintana-, and Bartonella koehlerae-specific antibodies using endpoint in-house immunofluorescence antibody assays. Bartonella real-time PCR (qPCR) and conventional PCR (cPCR) were performed. In addition, cPCR followed by DNA sequencing was performed for other pathogenic organisms (Anaplasma, Babesia, Cytauxzoon, Ehrlichia, Hepatozoon, hemotropic Mycoplasma, and Theileria spp.). RESULTS: From 135 cats studied, 80.7% were seroreactive against at least one Bartonella species. Bartonella quintana, B. koehlerae, and B. henselae seroreactivity was 67.4, 77.0, and 80.7%, respectively. Substantial to almost perfect serological agreement was found between the three Bartonella species. Colony cats were more likely to be Bartonella spp.-seroreactive than owned cats. Moreover, cats aged ≤ 2 years were more likely to be Bartonella spp.-seroreactive. Bartonella spp. DNA was detected in the blood of 11.9% (n = 16) of cats. Cats were infected with B. henselae (n = 12), B. clarridgeiae (n = 3), and B. koehlerae (n = 1). Mycoplasma spp. DNA was amplified from 14% (n = 19) of cat blood specimens. Cats were infected with Mycoplasma haemofelis (n = 8), Candidatus M. haemominutum (n = 6), Candidatus Mycoplasma turicensis (n = 4), and Mycoplasma wenyonii (n = 1). Anaplasma, Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria spp. DNA was not amplified from any blood sample. Of the 16 Bartonella spp.-infected cats based on PCR results, six (37%) were co-infected with Mycoplasma spp. CONCLUSIONS: Bartonella spp. and hemoplasma infections are prevalent in cats from the Barcelona area, whereas infection with Anaplasma spp., Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria infections were not detected. Co-infection with hemotropic Mycoplasma appears to be common in Bartonella-infected cats. To our knowledge, this study is the first to document M. wenyonii is infection in cats.

Bartonella henselae Antibodies in Serum and Oral Fluid Specimens from Cats.

Cats are the primary reservoir host for Bartonella henselae(B. henselae), an etiological agent of human bartonellosis, including cat scratch disease. Although Bartonella DNA has been amplified from salivary swabs from cats, dogs and humans, we are not aware of studies investigating Bartonella antibodies in oral fluid (OF). Using inhouse and commercial immunofluorescence antibody assays (IFA), the objective of this study was to detect and compare antibodies against B. henselae in paired OF and serum specimens from cats. Specimens were collected from shelter and client-owned cats. For serum specimens, B. henselae seroreactivity was 78% for both the inhouse and commercial IFA assays and 56.8% for OF specimens. Comparing serum and OF specimens, there was moderate Kappa agreement (Cohen's k = 0.434) for detection of B. henselae antibodies. Oral fluid antibodies were more likely measurable in cats with high B. henselae serum antibody titers when compared with low antibody titers. In conclusion, B. henselae OF IFA antibody measurements were less sensitive compared to serum IFA measurements of ≥1:64. Oral fluid antibodies were detected more often in cats with high B. henselae serum antibody titers. Therefore, OF antibodies, detectable by IFA, is of limited utility for epidemiological or diagnostic testing in cats.

The Effects of Polyhexamethylene Biguanide (PHMB) and TLR Agonists Alone or as Polyplex Nanoparticles against Leishmania infantum Promastigotes and Amastigotes.

Dogs are the main reservoir for Leishmania infantum, manifesting from a subclinical to a fatal disease. Limited treatments are available, although new antiparasitics and immunomodulators are pursued. Polyhexamethylene biguanide (PHMB) has a broad antimicrobial spectrum, including antiparasitic activity. Here, we evaluated the potential for Toll-like receptor agonists (TLRa) and PHMB alone, and as polyplex nanoparticles containing PHMB and TLR4 or TLR9 agonists, to selectively kill L. infantum. Susceptibility of L. infantum promastigotes to PHMB, miltefosine, and allopurinol was performed, and the half-maximum inhibitory concentrations (IC50) were determined. Then, DH-82 cells were infected and treated with PHMB alone or combined with TLR4a (MPLA-SM) or TLR9a (CpG ODNs) and allopurinol alone. The IC50 values of L. infantum promastigotes were PHMB (1.495 µM), miltefosine (9.455 µM), and allopurinol (0.124 µM). After infection, treated DH-82 cells displayed a lower percentage (p = 0.0316), intensity (p = 0.0002), and index of infection (p = 0.0022) when compared to non-treated cells. PHMB induced lower percentage of infection alone (p = 0.043), in combination with TLR9a (p = 0.043), and with TLR4a (p = 0.0213). Supernatants were collected and used to measure TNF-α and IL-6 levels. Increased TNF-α was observed after PHMB plus TLR4a, relative to uninfected and infected untreated macrophages (p = 0.043). PHMB combined with TLR4a shows promise as a potential anti-L. infantum drug combination, as well as inducer of proinflammatory response, as demonstrated by decreased infection and increased TNF-α production.

Immunotherapy in clinical canine leishmaniosis: a comparative update.

Leishmaniosis due to Leishmania infantum is a complex infection that can affect both humans and dogs, and present a wide range of clinical signs and clinicopathological abnormalities. The conventional treatment of this disease is challenging due to the fact that complete parasitological cure commonly does not occur. Furthermore, treatment of the disease with the conventionally used drugs has several shortcomings. These include the need for long-term treatment, side effects and the formation of drug resistance. Moreover, it is important to highlight that the host immune responses play a crucial role in the outcome of this infection. For this reason, the use of immunotherapy in clinical leishmaniosis to improve the result of treatment with the conventional anti-leishmanial drugs by enhancing the immune response is imperative. The aim of this review is to provide a comparative overview of the wide range of immunotherapeutical approaches and strategies for the treatment of L. infantum infection in animals focusing on dogs.

Toll-like receptors 2, 4 and 7, interferon-gamma and interleukin 10, and programmed death ligand 1 transcripts in skin from dogs of different clinical stages of leishmaniosis.

BACKGROUND: Canine leishmaniosis (CanL) caused by Leishmania infantum can have several dermatological manifestations. The type of immune response elicited against the parasite appears to be at the basis for such clinical variability. Much of the work in CanL has focused on adaptive immune response and there are scarce data on the importance of the innate immune responses. Moreover, few studies have evaluated the immunological response in the cutaneous lesions in dogs naturally infected with L. infantum and with different degrees of disease severity, and no study has compared clinically-lesioned with normal-looking skin. METHODS: We determined and compared the transcription of toll like receptors (TLRs) 2, 4 and 7, interferon gamma (IFN-γ), interleukin (IL) 10 and programmed cell death protein ligand (PD-L) 1 by real-time PCR in paired clinically-lesioned and normal-looking skin from 25 diseased dogs (mild disease-stage I (n = 11) and moderate to severe disease-stages II and III (n = 14) as well as in normal-looking skin from healthy dogs (n = 10) from a non-endemic area. We also assessed the association between the transcripts in clinically-lesioned and normal-looking skin of dogs with leishmaniosis with clinicopathological, immunological and parasitological findings. RESULTS: Clinically-lesioned skin from mildly affected dogs was characterized by a significant upregulation of TLR2 (P < 0.0001) and IL-10 (P = 0.021) and downregulation of TLR7 (P = 0.004) when compared with more severely affected dogs. Normal-looking skin of mildly affected dogs was characterized by a significant lower expression of TLR7 (P = 0.003), IFN-γ (P < 0.0001) and PD-L1 (P = 0.001) when compared with more severely affected dogs. TLR2, TLR4, IL-10 and IFN-γ upregulation in clinically-lesioned skin was correlated with lower disease severity while TLR7 upregulation was correlated with markers of disease severity. Upregulation of TLR7, IL-10, IFN-γ and PD-L1 in normal-looking skin was correlated with disease severity. CONCLUSIONS: This study demonstrated different expression profiles of immune genes in clinically-lesioned and normal-looking skin among mildly and more severely affected dogs. These immunological conditions might favor the maintenance and replication of the parasite in the skin of more severely affected dogs.

Total serum IgD from healthy and sick dogs with leishmaniosis.

BACKGROUND: Canine leishmaniosis (CanL) due to Leishmania infantum is characterized by the development of both cellular and humoral immune responses. The dysfunction of T cell-mediated immunity leads to a lack of proliferation of T cells in response to Leishmania antigens with the consequence of parasite dissemination that seems to be related to a T cell exhaustion mediated by regulatory B cells expressing immunoglobulin D (IgD). The aim of this study was to determine and compare the total serum IgD in dogs with clinical leishmaniosis and in clinically healthy dogs. RESULTS: A total of 147 dog sera were studied. All dogs were tested for L. infantum-specific antibodies by quantitative ELISA. Interferon-gamma (IFN-γ) production was also determined by sandwich ELISA after blood stimulation with L. infantum soluble antigen (LSA) or concanavalin A (ConA). The quantification of total IgD was performed using a human IgD sandwich ELISA quantification set. Dogs were classified in three different groups. Group 1 included 40 clinically healthy non-infected dogs, all serologically negative to L. infantum-specific antibodies and non-producers of IFN-γ upon LSA stimulation. Group 2 included 63 clinically healthy infected dogs that were LSA IFN-γ producers (n = 61) and/or IFN-γ non-producers (n = 2) as well as negative to medium seropositive to L. infantum antigen. Finally, Group 3 included 44 dogs with clinical leishmaniosis (IFN-γ producers, n = 23; and IFN-γ non-producers, n = 21) that were negative to highly positive to L. infantum-specific antibodies. No significant differences were observed when the total IgD concentration was compared within groups. Additionally, total IgD of sick IFN-γ producers and IFN-γ non-producers was not significantly different. Finally, total IgD concentration was not statistically related to demographic parameters such as age, sex and breed. CONCLUSIONS: The results of this study demonstrated that there were no differences between groups in total serum IgD. Total serum IgD does not appear to be a marker of disease in CanL.

Does co-infection with vector-borne pathogens play a role in clinical canine leishmaniosis?

BACKGROUND: The severity of canine leishmaniosis (CanL) due to Leishmania infantum might be affected by other vector-borne organisms that mimic its clinical signs and clinicopathological abnormalities. The aim of this study was to determine co-infections with other vector-borne pathogens based on serological and molecular techniques in dogs with clinical leishmaniosis living in Spain and to associate them with clinical signs and clinicopathological abnormalities as well as disease severity. METHODS: Sixty-one dogs with clinical leishmaniosis and 16 apparently healthy dogs were tested for Rickettsia conorii, Ehrlichia canis, Anaplasma phagocytophilum and Bartonella henselae antigens by the immunofluorescence antibody test (IFAT) and for E. canis, Anaplasma spp., Hepatozoon spp., Babesia spp. and filarioid DNA by polymerase chain reaction (PCR). RESULTS: Among the dogs examined by IFAT, the seroprevalences were: 69% for R. conorii, 57% for E. canis, 44% for A. phagocytophilum and 37% for B. henselae; while the prevalences found by PCR were: 8% for Ehrlichia/Anaplasma, 3% for Anaplasma platys and 1% for H. canis. No other pathogen DNA was detected. Statistical association was found between dogs with clinical leishmaniosis and seroreactivity to R. conorii antigen (Fisher's exact test: P = 0.025, OR = 4.1, 95% CI = 1-17) and A. phagocytophilum antigen (Fisher's exact test: P = 0.002, OR = 14.3, 95% CI = 2-626) and being positive to more than one serological or molecular tests (co-infections) (Mann-Whitney test: U = 243, Z = -2.6, n 1 = 14, n 2 = 61, P = 0.01) when compared with healthy dogs. Interestingly, a statistical association was found between the presence of R. conorii, E. canis, A. phagocytophilum and B. henselae antibodies in sick dogs and some clinicopathological abnormalities such as albumin and albumin/globulin ratio decrease and increase in serum globulins. Furthermore, seroreactivity with A. phagocytophilum antigens was statistically associated with CanL clinical stages III and IV. CONCLUSIONS: This study demonstrates that dogs with clinical leishmaniosis from Catalonia (Spain) have a higher rate of co-infections with other vector-borne pathogens when compared with healthy controls. Furthermore, positivity to some vector-borne pathogens was associated with more marked clinicopathological abnormalities as well as disease severity with CanL.